Glucose-stimulated insulin secretion occurs as a result of, and in proportion to, the rate of glucose metabolism in beta-cells of the islets of Langerhans. This proposal seeks to utilize techniques of molecular biology to alter the rate and regulation of glucose metabolism in clonal cells derived from islet and non-islet neuroendocrine tissues. We will evaluate the effects of altering several key metabolic regulatory activities in the AtT-20ins cell line system, and in parallel, in the rat insulinoma cell line RIN 1046-38 or in glucose unresponsive primary islet cells isolated from male Zucker Diabetic Fatty (ZDF) rats. The specific aims of the grant are as follows: 1) to test whether GLUT-2 and GLUT-1 have distinct effects on glucose-stimulated insulin release in islet cells or cell lines, as they clearly do in AtT-20ins cells, 2) to examine the effects of altering glucose phosphorylating capacity and kinetics by overexpressing native and mutant forms of glucokinase, and by reducing the level of expression of hexokinase in insulin secreting clonal cells, and 3) to examine the effects of increasing phosphofructokinase activity in insulin secreting clonal cells by overexpression of native and phosphatase deficient mutants of the fructose-2,6-kinase/fructose-2,6-- bisphosphatase enzyme. Alterations of glucose phosphorylation and phosphofructokinase will be studied in the presence and absence of GLUT-2 or GLUT-1 overexpression. The consequences of all of the molecular manipulations listed above will be examined at two levels, 1) the metabolic fate of glucose, measured by administration of strategically labeled glucose substrates (i.e., 2-, 3-, or 5-3H glucose) and by measurement of accumulation of certain metabolic end products (glycogen, lipids), and 2) the glucose-stimulated insulin secretion response. One long term goal of this research understand the metabolic consequences of manipulation of key proteins involved in regulation of carbohydrate metabolism in eucaryotic cells. A benefit of the systems chosen for this work (AtT-20ins and islet cells and cell lines) is that a second major goal can be pursued simultaneously, namely, the development of a clonal cell that faithfully mimics the glucose-stimulated insulin secretion function of islet beta-cells. Our hope is that such cells can be used in the future in a therapeutic strategy for insulin-dependent diabetes mellitus (IDDM), involving implantation of appropriately encapsulated engineered cells for cell-based insulin delivery in response to variations in circulating glucose.